Page 20
THE VELIGER
Vol. 6; Supplement
half of the ctenidium do not noticeably alter chemore¬
result in appreciable change of chemoreceptive abilities
ception. The osphradium functions as an olfactory organ
of T. funebralis.
and is capable of detecting the presence of an extract
containing 2.6 X 10“ parts by wet weight of tube feet
LITERATURE CITED
from Pisaster ochraceus. The osphradium, when stimu¬
lated, seems to cause the animals to be more sensitive
FEDER, H.
to stimulations of the head tentacles and epipodial struc¬
1956. Natural history of Pisaster ochraceus. Doctoral diss.,
tures. Removal of part of the osphradium does not
Stanford Univ.
Identification and Location of Carbohydrases in the
Intestinal Tract of Tegula funebralis
(Mollusca : Gastropoda)
WARREN R. BERRIE
AND
MITCHEL W. DEVEREAUX
Hopkins Marine Station of Stanford University.
Pacific Grove, California
(6 Tables)
SINCE Tegula funebralis (A. ADAMS, 1854) is an herbi-
parts: buccal cavity, salivary glands, esophagus, stomach
vorous marine animal, it is quite evident that it must
and digestive gland (since it was impossible to separate
have an efficient and well developed carbohydrate diges
the stomach from the digestive gland), digestive gland
tive mechanism. Although this subject has been explored
(portions freed from the stomach), spiral caecum, thin
to some degree already (GALLI, 1956), there still seemed
hindgut, and thick hindgut.
much to be investigated. We limited our efforts to the
The purpose of the first experiment was to determine
study of five carbohydrates present in the environment
the site of enzyme production. The gut segments were
of the animal: starch, laminarin, alginate, fucoidin, and
excised from snails fresh from the field. These tissues
cellobiose. Using these materials we hoped to localize the
were refrigerated so as to retard any loss of enzyme
points of enzyme production within the alimentary canal
activity due to denaturation or autolysis. Pools of tissue
and sites of carbohydrate digestion. The extent to which
from five animals were washed, weighed, and extracted
intracellular and extracellular digestion is involved in the
in a tissue grinder equipped with a Teflon pestle (Van
foregut and hindgut was also studied.
Waters and Rogers Inc., Catalogue no. 48652) with either
The substrates were: starch (Baker and Adamson,
a citrate-phosphate buffer, pH 5.8, or a phosphate buffer.
reagent grade), alginate (Kelco Co., commercial grade),
pH 7.4. These buffers were chosen since they approximate
cellobiose (Pfanstiehl Chemical Co., C. P grade). Lamin¬
the extremes of hydrogen ion concentration occurring in
arin and fucoidin were isolated from Fucus by the
the digestive tract. Enzyme activity was determined by
method of BLACK, DEWAR, & WOODWARD (1951 - 1952).
incubating an appropriate aliquot of tissue extract with
The alimentary canal was divided into eight anatomical
one of the substrates and assaying for reducing sugar by
Page 21
Vol. 6; Supplement
THE VELIGER
the Somogyi reaction as modified by GALLI (1956).
Table 2
Enzyme and substrate controls were incubated simulta¬
Enzyme Controls
neously. Incubation was at 15° C for 24 hours.
Tissue extracts (w/v) : buccal cavity, 0.42% ; salivary
The results are recorded in Tables 1, 2, and 3. It
glands, 0.03%; esophagus, 0.06%; stomach and digestive
appears that throughout the entire length of the digestive
gland, 0.21% ; digestive gland, 0.04%; spiral caecum,
tract there is widespread production of large concentra¬
0.05%; thin hindgut, 0.06%; thick hindgut, 0.07%;
tions of amylase and cellobiase. On the other hand,
citrate-phosphate buffer, 0.05M, pH 5.8; phosphate
there was only a small and localized production of
buffer, O.1M, pH 7.4; incubated at 15° C for 24 hours.
enzymes that split the 1,4-ß-p-mannuronic acid linkage
of algininic acid, the 1,2-a-L-fucose-4-sulfate linkage of
uug Reducing Sugar'
Tissue
fucoidin, and the 1,3-ß-n-glucose linkage of laminarin.
pH 5.8
The results would further indicate that the salivary glands
buccal cavity
19.0
do not play an important role in enzyme production
with the exception of cellobiase. It is also evident from
salivary gland
92.3
Table 3 that some of the enzymes show different pH
esophagus
19.5
stomach and
optima.
digestive gland
digestive gland
Table 1
spiral caecum
18.0
Substrate Controls
thin hindgut
Substrates, 0.1%; citrate-phosphate buffer, 0.5M, pH 5.8;
thick hindgut
82.0
phosphate buffer, O.1M, pH 7.4; incubation at 15° C
pH 7.4
for 24 hours.
48.5
buccal cavity
ug Reducing Sugar
Substrate
salivary gland
esophagus
pH 5.8
stomach and
70.4
starch
digestive gland
laminarin
digestive gland
21.0
alginate
spiral caecum
fucoidin
thin hindgut
312.0
cellobiose
thick hindgut
pH 7.4
reducing sugar present in 1 mg dry weight of tissue.
starch
laminarin
buccal cavity through the mouth, and into the thick
alginate
hindgut through the anus. The ligated snails were held at
fucoidin
15° C for 24 hours submerged in Millipore filtered sea
474.0
cellobiose
water. The snails survived this period of incubation. At
ug reducing sugar present in 1 ml substrate
the end of the incubation period, the ligated areas were
excised, homogenized, and assayed for reducing sugar.
A duplicate experiment was performed in which a
The second experiment was conducted to investigate
mixture of antibiotics was incorporated with the substrate
the site of enzyme action. The snails were starved unti
in order to assess the role played by intestinal bacteria in
the digestive tract was cleared of material. A period of
carbohydrate hydrolysis. The antibiotic mixture con¬
about ten days was required. Ligatures were used to
tained: 50 units/ml penicillin, 25 ug/ml streptomycin,
isolate the previously mentioned parts of the digestive
25 ug/ml terramycin, 5 ug/ml polymixin
tract.
The results of these experiments are shown in Tables
The salivary glands were cut away since they could not
4 and 5. A comparison of Tables 3 and 4 reveals some
be excluded by ligatures. It was impossible to make any
further points of interest. The salivary glands and esoph-
type of ligation between the stomach and digestive gland.
agus contain a large amount of cellobiase; yet the absence
Ligatures were of nylon thread. Substrate was injected
of cellobiose hydrolysis in the esophagus suggests that
through the wall into the ligated part with the aid of a
26-gauge needle. The substrate was injected into the
the enzymes do not act upon the glucose-B-1,4-glucose
Page 22
Vol. 6; Supplement
THE VELIGER
Table 3
Reducing Sugar Released by Enzyme Action
Substrates, 0.1%; citrate-phosphate buffer, O.05M,
pH 5.8; phosphate buffer, O.1M, pH 7.4; incubation at
15° C for 24 hours.
Anatomical Region
Substrates
Starch Laminarin Alginate Fucoidin Cellobiose
ug Reducing Sugar
pH 5.8
buccal cavity
52.5
136.6
53.1
119.0
salivary gland
1988.0
esophagus
49.5
207.
872.7
1008.5
stomach and
322.5
38.5
341.0
digestive gland
digestive gland
131.5
spiral caecum
1206.0
105.0
2310.7
406.0
thin hindgut
1888.0
thick hindgut
1698.0
63.4
61.0
46.5
1953.0
pH 7.4
buccal cavity
430.5
161.4
183.3
212.5
salivary gland
746.0
esophagus
107.6
293.7
stomach and
525.6
529.6
digestive gland
digestive gland
2164.9
113.0
834.0
spiral caecum
2142.0
thin hindgut
710.0
1504.0
thick hindgut
689.4
434.8
ug reducing sugar released by enzyme extracted from 1 mg dry weight
of tissue. The values in the table have been corrected by subtraction of
substrate and enzyme controls (see tables 1 and 2).
linkages in this portion of the digestive tract. A compar¬
in the lumen or in the wall of the foregut and hindgut.
Carbohydrates were again injected into the various seg
ison of Tables 4 and 5 shows that antibiotics decrease
ments after ligation, and the snails were incubated for 24
carbohydrate hydrolysis and suggests that microorganisms
are, in part, involved in the breakdown of carbohydrates
hours. The areas were excised, opened, and washed with
in all parts of the digestive tract. Hydrolysis of starch and
distilled water. Both the washings and a homogenate of
cellobiose was most markedly affected by antibiotics.
the tissue were then tested for reducing sugar.
The results presented in Table 6 suggest that the major-
Bacteria were cultured from the foregut and hindgut.
The medium used contained: 1% agar, 0.001% aqueous
ity of cellobiose hydrolysis in the hindgut occurred in the
wall. Such a simple interpretation is not possible. The
extract of snail, 0.001% yeast extract, 0.01% Difco pep¬
test procedure only determined the amounts of reducing
tone, and 0.05% of one of the carbohydrate substrates
and was buffered at one of the two different pH's — 5.8
sugar present in lumen and tissue. Cellobiose is a reducing
or 7.4. A variety of colonial types developed. Hydrolysis
sugar itself. An adequate correction for the reducing
activity of the substrate when it is possibly divided
of starch by the organisms was detected by loss of a
between tissue and lumen is not possible. Since hydrolysis
reaction with iodine. The presence of reducing sugars
of this disaccharide does not result in a great change in
could be demonstrated in the other cultures by Tollen's
molecular size, no marked difference in the absorption of
Reagent. Splitting of cellobiose could not be determined
the substrate and products of its hydrolysis might be
by these methods.
The purpose of the final set of experiments was to
expected. No distinction between intracellular digestion
and absorption of the products of digestion can be made
determine whether carbohydrate hydrolysis was occurring
Page 23
Vol. 6; Supplement
THE VELIGER
Table 4
Reducing Sugars Released in Situ
Substrates, 0.1% ; incubation at 15°C for 24 hours
Substrates
Anatomical Region
Starch Laminarin Alginate Fucoidin Cellobiose
ug Reducing Sugar
193.5
1965.0
240.0
220.3 321.5
buccal cavity &
salivary gland
155.5
113.5
170.0
buccal cavity
249.5
182.3
esophagus
105.5
103.4
65.7
282.6 521.6 1032.6
stomach and
873.6
digestive gland
2680.0
296.0 289.2
1113.0
849.3
spiral caecum
132.0
1772.8
thin hindgut
416.0
152.7 172.1
1594.7
thick hindgut
1660.0
63.9
ug reducing sugar corrected by subtraction of appropriate tissue and
substrate controls.
Table 5
Reducing sugar released in Situ in the Presence of
an Antibiotic
Substrates, 0.1% ; incubation at 15° C for 24 hours.
Substrates
Anatomical Region
Starch Laminarin Alginate Fucoidin Cellobiose
ug Reducing Sugar
1760.4
180.6
190.0
buccal cavity &
253.7
salivary gland
87.5 145.0
buccal cavity
107.5
0
esophagus
169.3
stomach and
184.6
155.8
digestive gland
132.6
466.0
228.5
spiral caecum
208.5
135.6 242.0
120.8 154.0
430.7
thin hindgut
307.0
112.2
63.9
14130
thick hindgut
ug reducing sugar corrected by subtraction of appropriate tissue and sub-
strate controls.
and are then carried to the hindgut in an active form.
in the case of this small molecular weight substrate. The
GALLI (1956), evidently, did not expect the hindgut to
results with starch, on the other hand, do indicate
be important in digestion since he ignored this organ in
appreciable intracellular digestion in the hindgut tissue.
his studies. Yet the activity of the hindgut was clearly
demonstrated in our experiments. Also, when GALLI
DISCUSSION
studied the foregut, he excluded the esophagus and con-
The observed enzymatic activity of the hindgut has not
centrated on the buccal cavity and salivary glands. This
been previously reported. Both a cellobiase and an amyl-
was unfortunate since our results showed a reasonably
ase are present here. Specific enzymes capable of hydro¬
wide range of activity here. All the carbohydrates tested
underwent some decomposition in the esophagus, also
lyzing the other substrates tested also appear to be present
in this part of the gut. However, it appears likely that
Table 3 indicates that some of the enzymes may be
the other enzymes are produced in the foregut or midgut
produced here.
Vol. 6; Supplement
THE VELIGER
Page 24
Table 6
Distribution of Reducing Sugar Formed in Situ
Substrates: starch, laminarin, alginate, fucoidin, 0.1%
cellobiose, 0.15% ; incubation at 15° C for 24 hours.
ug Reducing Sugar Present in Lumen
Substrates
Anatomical Region
Starch Laminarin Alginate Fucoidin Cellobiose
ug Reducing Sugar
172.3
87.4
256.9
321.7
foregut
324.0
68.6
13.1
hindgut
ug Reducing Sugar Present in Wall
Substrates
Anatomical Region
Starch Laminarin Alginate Fucoidin Cellobiose
ug Reducing Sugar"
foregut
684.5
hindgut
152.8
0
corrected by subtraction of substrate control.
corrected by subtraction of tissue control.
SUMMARY
LITERATURE CITED
Carbohydrate digestion in the snail Tegula funebralis
BLACK, W. A. P., W. J. CORNHELL, E. T DEWAR & E N. WOODWARD
(A. ADAMS, 1854), was studied. The intestinal sites of
1951. Manufacture of algal chemicals: Laboratory-scale iso¬
hydrolysis of certain carbohydrates and the location of
lation of Laminarin from brown marine algae. Journ. appl.
production of their respective enzymes were studied.
Chem. 1: 505 -517
The presence of an amylase, laminarase, alginase, fuco¬
BLACK, W. A. P., E. T. DEWAR & F N. WOODWARD
idase, and cellobiase was demonstrated. The major tissue
1952. Manufacture of algal chemicals: Laboratory-scale iso¬
sources of the above enzymes were: buccal cavity, stom¬
lation of Fucoidin from brown marine algae. Journ. Sci.
of Food and Agricult. 3: 122 - 129
ach, digestive gland, spiral caecum, thin and thick hindgut
for amylase; buccal cavity and esophagus for laminarase
GALLI, DONALD RICHARD
and alginase; buccal cavity, esophagus, stomach and
1956. Carbohydrate digestion in a herbivorous marine snail,
digestive gland for fucoidase; salivary glands, spiral
Tegula funebralis. Master of Arts Thes., Stanford Univ.; 153
pages
caecum, thin and thick hindgut for cellobiase. Some of
the carbohydrate hydrolysis appears to be due to bacterial
action. Intracellular digestion of starch in the wall of the
hindgut is indicated.
Page 25
Vol. 6; Supplement
THE VELIGER
A New Pigment from Tegula funebralis
(Mollusca: Gastropoda)
PATRICIA MCGEE
Hopkins Marine Station of Stanford University,
Pacific Grove, California
(1 Text figure; 3 Tables)
change. Prolonged heating at 100° C resulted in a brown
IN THE TROCHACEAN SPECIES Tegula funebralis (A.
discoloration, and is assumed to be due to decomposition.
ADAMS, 1854) and T. brunnea (PHILIPPI, 1848) which
It is photosensitive, and becomes yellow with prolonged
are abundant in the intertidal zone of the Pacific coast,
exposure to light. When reduced with hydrosulfite, a yel-
the female gonad is bright green. In both species, the
low color appears, but reoxidation to green can be achieved
pigment is found in droplets evenly dispersed throughout
by autoxidation or treatment with H.O2. In methanol,
the volk. An extraction of the pigment in T. funebralis
a green fluorescence was observed in the oxidized form.
was made. The crude green pigment was partitioned into
The large molecular size suggested a protein complex.
a group of yellow carotenoids and an unknown green
The unknown was, therefore, placed in aqueous solution
pigment. This is a study of these colored materials.
and an equal amount of CHCIs with .1 volume amylal¬
PREPARATION
cohol added. This was vigorously shaken and centrifuged
for 10 minutes. A blue protein appeared between the
The pigment was initially extracted from eggs carefully
phases of green aqueous solution and colorless CHCIa.
stripped from 200 gonads. The eggs were blended in a
The aqueous phase was re-treated until the blue zone no
Waring blender with absolute methanol for five minutes.
longer appeared. The solubilities and spectrum of the un-
The methanol was changed, and extraction was repeated
until the suspension was white. The crude green pigment
was then dried and redissolved in methanol. This re¬
RELATIVE ABSORPTION
extraction was repeated three times. Ether and water
partition of the pigment separated the material into a
vellow epiphase and a green hypophase. Repeated parti-
07
tioning was used to purify the materials.
RESULTS
The yellow material, dried and dissolved in petroleum
ether (Bp. 40° 60° C) was placed on an Al-Oa column.
The column was developed with a gradient of acetone in
petroleum ether. The four bands observed were collected,

and a tentative identification of zeaxanthin, lutein and
alpha carotene was made from the data in Table I (P.

KARRER & E. JUCKER, 1950).
The green pigment could not be identified. Some of its
250 300 350 400 450 500 550 600 650 700
properties are briefly stated in Table II. In addition, in
aqueous solution it freely passed through sephadex G-75
which indicates a molecular size larger than that cor-
Figure 1: The absorption maxima of the unknown green
responding to a molecular weight of 40,000. In aqueous
pigment in H.O. The oxidized state peaks at 640 and 273,
solution, it may be warmed to 100° C without obvious
while the reduced state peaks at 273 u.
Page 26
Vol. 6; Supplement
THE VELIGER
Table 1
PROPERTIES OF YELLOW PIGMENTS
ABSORPTION
CAROTENOID“
BAND MAXIMA in CS.
REMARKS
I 517 482 450
hypophasic in petroleum
zeaxanthin
ether and 90% MeÖH
II 510 470 442
distributed in both
phases of pet. ether
and 90% MeOH
III 508 475 445
hypophasic in petroleum
lutein
ether and 90% MeÖH
a - carotene
509 477
epiphasic in petroleum
ether and 90% MeÖH
* Tentative identification on the basis of the
absorption spectra and solubility properties.
Table 2
PROPERTIES OF GREEN PIGMENT
COLORATION
ABSORPTION MAXIMA mu
SOLUBILITY
PIGMENT
reduced
reduced
oxidized
oxidized
neutral¬
neutral¬
H.O
s. H.O, MeOH,
H.O
1. Native
yellow
green
EtOH, acet.
640 273
273
acid¬
acid¬
eth., CHCI,
MeoH
vellow
vellow
640 370*(s)
pet. eth.,
alkaline-
alkaline-
CS.
vellow
yellow
with ppt.
same as 1
same as 1
same as 1
same as 1.
same as 1
except
Deproteinized
alkaline-
vellow
with no
ppt.
neutral¬
neutral¬
H.O
H.O
s. H.O
yellow-
Allagochrome
630 320 260
320 260
green
acid¬
orange
(Habermann,
acid¬?
1960)
alkaline-
alkaline-?
brown
* (s) = shoulder.
s. = soluble
i. = insoluble
eth. = ether
pet. eth. = petroleum ether
acet. — acetone
Page 27
Vol. 6; Supplement
THE VELIGER
is that coenzyme Q is necessary for respiratory electron
Table 3
transfer occurring in the mitochondria and equivalent
Results of mass spectral analysis of crude
structures. While several quinones of biological origin have
green pigment in percent by weight
been described, their actual involvement in electron trans-
ELEMENT PERCENT ELEMEN
PERCENT
port has seldom been demonstrated. If a respiratory func-
tion for the unknown pigment is postulated, the observed
0.005
Al
migration of the pigment to the ciliated cells of the velum
Ca
0.01
0.10
of the trochophore and veliger larvae may be of signif-
Cu
0.005
0.005
icance. These cells would be expected to have a high
0.01
0.01
metabolic activity.
Mg
0.03
Zn
trace
Mn
trace
SUMMARY
known without protein appear in Table II. It should be
The pigments of the eggs of Tegula funebralis were ex¬
tracted in methanol. This crude green pigment contained
noted that the absorption spectrum of the oxidized form
three yellow materials with spectral properties resembling
was not changed.
the carotenoids: zeaxanthin, lutein and alpha carotene,
In preliminary observations of the development of the
and an unknown green pigment. The green pigment was
closely related Tegula brunnea the green pigment present
found to have an attached protein, absorption maxima
in the eggs was observed to concentrate in the ciliated
in H.O at 640, 273mu in the oxidized form and 273 mu
cells of the trochophore. The amount appeared to in¬
in the reduced state, and a marked resemblance to known
crease as the velum formed.
quinones that have been suggested to act as respiratory
DISCUSSION
pigments.
The unknown pigment resembles Allagochrome (HABER-
LITERATURE CITED
MANN, 1960; GARRICK & HABERMANN, 1962) in its ab¬
CRANE, F. L.
sorption spectrum and oxidation-reduction activities. A
1959.
Internal distribution of Coenzyme Q in higher plants.
comparison of the two pigments' properties can be seen
Plant Physiol. 34: 128 - 131
in Table II. Allagochrome is present in a variety of higher
GARRICK, L. S. & H. M. HABERMANN
plants and its function is hypothesized to be respiratory
1962. Distribution of allagochrome in vascular plants.
due to the ease with which oxidation and reduction can
Amer. Journ. Bot. 49: 1078 -1088
be induced
HABERMANN, H. M.
The peak at 273 mu also is suspiciously near the char-
1960. A new leaf pigment (pp. 73 -82 in:) Comp. Biochem.
acteristic peak, 275 mu of the coenzyme Q, a lipid soluble
of photoreactive systeins; Acad. Press, New York and London.
quinone (CRANE, 1959). There is a broad distribution of
xii + 437 pp.
the five known forms of coenzyme Q in aerobic tissues
KARRER, P & E. JUCKER
It has been found in all vertebrates, higher plants, aerobic
1950. Caretonoids. Elsevier Publ. Co., Inc. New York etc.
bacteria, invertebrates and red and green alga. The view
x + 384 pp.